首页> 外文OA文献 >Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.
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Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

机译:荧光标记的牛酰基CoA结合蛋白,可作为酰基CoA传感器:与CoA和酰基CoA酯的相互作用及其在测量游离酰基CoA酯和非酯化脂肪酸中的用途。

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摘要

Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.
机译:长链酰基辅酶A酯是脂质合成和β氧化的关键代谢产物,但同时也是中间代谢,胰岛素分泌,囊泡运输和基因表达的重要调节剂。研究酰基辅酶A酯的调节功能的关键工具是测定游离酰基辅酶A浓度的可靠方法。目前尚无此类方法。在本研究中,我们描述了使用酰基辅酶A结合蛋白作为支架来测量游离酰基辅酶A浓度的两种酰基辅酶A传感器的合成。牛酰基辅酶A结合蛋白的Met24和Ala53被半胱氨酸残基取代,其被6-溴乙酰基-2-二甲基氨基萘共价修饰以制成两个荧光酰基辅酶A指示剂FACI-24和FACI-53。在不存在配体的情况下(激发387 nm),FACI-24和FACI-53分别在510和525 nm处显示最大荧光发射。用十六烷酰基-CoA和十二烷酰基-CoA滴定FACI-24和FACI-53,分别在460和495 nm处荧光产量提高了5.5倍和4.7倍。当结合C14-C20饱和和不饱和酰基-CoA酯时,FACI-24在460 nm处显示出高且相似的荧光产量增加。两种指示剂均以高特异性和亲和力(K(d)= 0.6-1.7 nM)结合长链(> C14)酰基辅酶A酯。 FACI-53对C8-C12酰基链显示出高荧光产量。结果表明,FACI-24可作为灵敏的酰基辅酶A传感器,用于测量脂肪酸转化为各自的酰基辅酶A酯后的游离酰基辅酶A浓度,酰基辅酶A合成酶活性和游离脂肪酸的浓度。 。

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